Immunofluorescent Staining of Lung Sections

Lung sections were stained using a modified protocol based on published reports (34 (link), 35 (link)). Five micrometer sections from paraffin-embedded lung biopsies from control or IPF patients were dewaxed prior to heat-induced epitope retrieval with Tris-EDTA buffer, pH 9.0. Sections were blocked with Fc block (BD biosciences, UK) before incubation with a blocking buffer (5% goat serum/2.5% BSA/PBS/0.1% Tween-20) for 1 h. Slides were then washed and incubated with anti-citrullinated histone H3 (Abcam, UK) and anti-MPO (R&D systems, UK) antibodies diluted in 0.5x blocking buffer overnight at 4° C. Anti-rabbit Alexa Fluor^® 647-conjugated and anti-mouse Alexa Fluor^® 555-conjugated secondary antibodies (Invitrogen, UK) and DAPI (Sigma, UK) diluted in 0.5x blocking buffer were then added for 30 min. Stained sections were washed, mounted, sealed and visualized using an Olympus inverted fluorescence confocal microscope and analyzed using Fluoviewer software (Olympus).